Total internal reflection fluorescence microscopy is a particular measurement method in light microscopy in which an excitation light beam is typically totally reflected at an interface between a cover glass or slide and a sample. This generates a so-called evanescent (decaying) field in the sample which may excite the sample to fluorescence. The penetration depth of the evanescent fields depends on the exact angle of incidence of the excitation light beam.
As the critical angle for total reflection, i.e. the threshold angle at which a transmitted light beam vanishes, depends on the indices of refraction of the materials involved, this angle depending on the type of samples used, may not be known from the start. Therefore, it is desirable to be able to determine the critical angle.
United States Patent Publication No. 2011/0057093 A1 discloses a method evaluating the intensity of an optical response of a sample depending on an angle of incidence of irradiation light. To increase robustness of the method, an analysis is performed in a pupil plane which sometimes requires additional optical elements.
Therefore, a need exists for methods and devices for determining such a critical angle which should be easy to implement, preferably without using additional optical elements, and which are robust.
Thus, a need exists to overcome the problems with the prior art systems, designs, and processes as discussed above.